ASTM F813-20

Standard Practice for Direct Contact Cell Culture Evaluation of Materials for Medical Devices

SIGNIFICANCE AND USE
4.1 This practice is useful for assessing cytotoxic potential both when evaluating new materials or formulations for possible use in medical applications, and as part of a quality control program for established medical materials and medical devices.  
4.2 This practice assumes that assessment of cytotoxicity potential provides one method for predicting the potential for cytotoxic or necrotic reactions to medical materials and devices during clinical applications to humans. In general, cell culture testing methods have shown good correlation with animal assays when only chemical toxicities are being considered.
Note 1: The results obtained using this method may not predict in vivo behavior which can be influenced by multiple factors such as those arising from site of application or physical properties that may result from design and fabrication.  
4.3 This cell culture test method is suitable for adoption in specifications and standards for materials for use in the construction of medical devices that are intended to have direct contact with tissue, tissue fluids, or blood. However, care should be taken when testing materials that are absorbable, include an eluting or degradable coating, are liquid or gelatinous in nature, are irregularly shaped solid materials, or have a high density or mass, to make sure that the method is applicable. If leachables from the test sample are capable of diffusing through the agar layer, agarose-based methods such as Test Method F895 may be considered as an alternate method, depending on sample characteristics, or in cases where investigators wish to further evaluate the cytotoxic response of cells underlying the test sample.
SCOPE
1.1 This practice covers a reference method of direct contact cell culture testing which may be used in evaluating the cytotoxic potential of materials for use in the construction of medical materials and devices.  
1.2 This practice may be used either directly to evaluate materials or as a reference against which other cytotoxicity test methods may be compared.  
1.3 This is one of a series of reference test methods for the assessment of cytotoxic potential, employing different techniques.  
1.4 Assessment of cytotoxicity is one of several tests employed in determining the biological response to a material, as recommended in Practice F748.  
1.5 The L-929 cell line was chosen because it has a significant history of use in assays of this type. This is not intended to imply that its use is preferred; only that the L-929 is a well characterized, readily available, established cell line that has demonstrated reproducible results in several laboratories.  
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 31-Mar-2020
• ICS: 07.100.10 - Medical microbiology
• Technical Committee: F04 - Medical and Surgical Materials and Devices
• Drafting Committee: F04.16 - Biocompatibility Test Methods

Relations

• Refers: ASTM F748-16 - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 01-Apr-2016
• Refers: ASTM F895-11(2016) - Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
• Effective Date: 01-Apr-2016
• Refers: ASTM F1027-86(2012) - Standard Practice for Assessment of Tissue and Cell Compatibility of Orofacial Prosthetic Materials and Devices
• Effective Date: 01-Oct-2012
• Refers: ASTM F895-11 - Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
• Effective Date: 01-Oct-2011
• Refers: ASTM F748-06(2010) - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 01-Jun-2010
• Refers: ASTM F1027-86(2007) - Standard Practice for Assessment of Tissue and Cell Compatibility of Orofacial Prosthetic Materials and Devices
• Effective Date: 01-Feb-2007
• Refers: ASTM F748-06 - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 01-Dec-2006
• Refers: ASTM F895-84(2006) - Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
• Effective Date: 01-Mar-2006
• Refers: ASTM F748-04 - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 01-May-2004
• Refers: ASTM F1027-86(2002) - Standard Practice for Assessment of Tissue and Cell Compatibility of Orofacial Prosthetic Materials and Devices
• Effective Date: 01-Jan-2002
• Refers: ASTM F748-98 - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 10-Aug-1998
• Refers: ASTM F895-84(2001) - Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
• Effective Date: 01-Jan-1995
• Refers: ASTM F895-84(2001)e1 - Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
• Effective Date: 01-Jan-1995

Overview

ASTM F813-20: Standard Practice for Direct Contact Cell Culture Evaluation of Materials for Medical Devices establishes a reference method for assessing the cytotoxic potential of materials intended for use in medical devices. This practice outlines procedures for direct contact cell culture testing, providing a critical evaluation tool for determining the biocompatibility of materials that will come into direct contact with tissue, tissue fluids, or blood.

This standard is widely used in the medical device industry as an essential part of material evaluation and quality control processes. It applies to both new and existing materials, helping manufacturers and researchers predict potential cytotoxic or necrotic reactions that may occur during clinical applications. While in vitro cell culture methods offer a rapid and effective means to screen for chemical toxicities, results may not always predict in vivo behavior due to additional factors such as device design or implantation site.

Key Topics

• Direct Contact Cell Culture Testing: Describes the methodology for placing test specimens in direct contact with a confluent layer of cultured cells (commonly the L-929 mouse fibroblast cell line), enabling sensitive detection of cytotoxic effects.
• Assessment Criteria: Provides a qualitative scheme for categorizing observed cytotoxic effects, ranging from no detectable response to severe cell damage extending beyond the area of contact.
• Control Materials: Prescribes the use of both positive and negative control materials in each assay to validate test results.
• Applicability and Limitations: Advises caution when testing absorbable materials, eluting or degradable coatings, liquids, gels, and irregularly shaped specimens, and discusses alternate methods such as agar diffusion or extraction techniques for special cases.
• Reporting Requirements: Specifies the information to be included in test reports, such as materials tested, cell line and culture conditions, sterilization method, and pass/fail outcomes.

Applications

Adoption of ASTM F813-20 supports medical device manufacturers, researchers, and regulatory authorities in the following ways:

• Material Selection: Helps in screening and selecting safe materials for medical devices that will have direct patient contact, reducing the risk of adverse cytotoxic reactions.
• Quality Control: Integrates into routine quality assurance programs to ensure consistency and safety of materials and finished devices.
• Regulatory Compliance: Provides a recognized and internationally accepted methodology that supports regulatory submissions and approvals.
• Comparison of Test Methods: Serves as a benchmark reference for evaluating and validating alternative cytotoxicity assays, enhancing reliability and reproducibility in biocompatibility testing.

By facilitating early identification of cytotoxic materials, this standard minimizes the risk of device failure or adverse patient outcomes, and helps streamline the product development lifecycle.

Related Standards

• ASTM F748 - Practice for Selecting Generic Biological Test Methods for Materials and Devices: Guidance for choosing appropriate biological assessment techniques.
• ASTM F619 - Practice for Extraction of Medical Plastics: Procedures for preparing extracts from device materials for testing.
• ASTM F895 - Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity: Alternate agarose-based assay for situations where direct contact may not be suitable.
• ASTM F1027 - Practice for Assessment of Tissue and Cell Compatibility of Orofacial Prosthetic Materials and Devices: Specialized assessment for prosthetic materials.
• USP Negative Control Plastic Reference Standard - Recognized negative control for validating cytotoxicity assays.

Keywords: biocompatibility, cell culture, cytotoxicity testing, direct contact assay, medical device materials, material safety evaluation, ASTM standards, L-929 cell line, biological response testing.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: F813 − 20
Standard Practice for
Direct Contact Cell Culture Evaluation of Materials for
Medical Devices
ThisstandardisissuedunderthefixeddesignationF813;thenumberimmediatelyfollowingthedesignationindicatestheyearoforiginal
adoptionor,inthecaseofrevision,theyearoflastrevision.Anumberinparenthesesindicatestheyearoflastreapproval.Asuperscript
epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2. Referenced Documents
1.1 Thispracticecoversareferencemethodofdirectcontact 2.1 ASTM Standards:
cell culture testing which may be used in evaluating the F748PracticeforSelectingGenericBiologicalTestMethods
cytotoxic potential of materials for use in the construction of for Materials and Devices
medical materials and devices. F895TestMethodforAgarDiffusionCellCultureScreening
for Cytotoxicity
1.2 This practice may be used either directly to evaluate
F1027Practice for Assessment of Tissue and Cell Compat-
materialsorasareferenceagainstwhichothercytotoxicitytest
ibility of Orofacial Prosthetic Materials and Devices
methods may be compared.
2.2 Other Documents:
1.3 This is one of a series of reference test methods for the
The American Type Culture Collection (ATCC), Catalogue
assessment of cytotoxic potential, employing different tech-
of Strains II
niques.
USP Negative Control Plastic Reference Standard
1.4 Assessment of cytotoxicity is one of several tests
employed in determining the biological response to a material, 3. Summary of Practice
as recommended in Practice F748.
3.1 Cell cultures are grown to a confluent monolayer in
1.5 The L-929 cell line was chosen because it has a culture dishes. The growth medium is aspirated and replen-
significant history of use in assays of this type. This is not ishedtoprovidearesting,confluentcelllayer.Testandcontrol
intended to imply that its use is preferred; only that the L-929 specimens are placed in direct contact with the cell layer to
is a well characterized, readily available, established cell line provide an accelerated assessment of the presence or absence
that has demonstrated reproducible results in several laborato- of a cytotoxic effect from a given material or device. See
ries. Practice F1027 for definitions.
1.6 The values stated in SI units are to be regarded as
4. Significance and Use
standard. No other units of measurement are included in this
4.1 This practice is useful for assessing cytotoxic potential
standard.
both when evaluating new materials or formulations for
1.7 This standard does not purport to address all of the
possible use in medical applications, and as part of a quality
safety concerns, if any, associated with its use. It is the
control program for established medical materials and medical
responsibility of the user of this standard to establish appro-
devices.
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use. 4.2 This practice assumes that assessment of cytotoxicity
1.8 This international standard was developed in accor- potential provides one method for predicting the potential for
cytotoxicornecroticreactionstomedicalmaterialsanddevices
dance with internationally recognized principles on standard-
ization established in the Decision on Principles for the during clinical applications to humans. In general, cell culture
testing methods have shown good correlation with animal
Development of International Standards, Guides and Recom-
mendations issued by the World Trade Organization Technical assays when only chemical toxicities are being considered.
Barriers to Trade (TBT) Committee.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Surgical Materials and Devices and is the direct responsibility of Subcommittee Standards volume information, refer to the standard’s Document Summary page on
F04.16 on Biocompatibility Test Methods. the ASTM website.
Current edition approved April 1, 2020. Published June 2020. Originally American Type Culture Collection, P.O. Box 1549, Manassas, VA 20108.
approvedin2001.Lastpreviouseditionapprovedin2012asF813–07(2012).DOI: U.S. Pharmacopeia, Vol 24, Rand McNally, Taunton, MA, 1994, pp.
10.1520/F0813-20. 1652–1653. Use latest publication to ensure current cumulative revisions are used.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F813 − 20
NOTE1—Theresultsobtainedusingthismethodmaynotpredict in vivo may, however, have an adverse effect on the viability of the cell cultures.
behaviorwhichcanbeinfluencedbymultiplefactorssuchasthosearising
6.1.2 L-glutamine Solution, 29.2 mg/mL of sterile water.
from site of application or physical properties that may result from design
6.1.3 Hanks’ Solution, calcium-and magnesium-free (store
and fabrication.
at room temperature).
4.3 This cell culture test method is suitable for adoption in
6.1.4 Trypsin,0.1%solutioninHanks’solutionorcalcium-
specifications and standards for materials for use in the
and magnesium-free, phosphate-buffered saline (store frozen).
constructionofmedicaldevicesthatareintendedtohavedirect
6.1.5 Water, distilled, deionized, and sterile, with a mini-
contact with tissue, tissue fluids, or blood. However, care
mum resistivity of 1 MΩ·cm.
should be taken when testing materials that are absorbable,
include an eluting or degradable coating, are liquid or gelati-
6.2 All reagents shall be tissue-culture grade or equivalent.
nous in nature, are irregularly shaped solid materials, or have
6.3 Reagents shall be reconstituted in accordance with the
a high density or mass, to make sure that the method is
manufacturer’s directions, using aseptic technique.
applicable. If leachables from the test sample are capable of
6.4 Reagents shall be stored in accordance with the manu-
diffusing through the agar layer, agarose-based methods such
facturer’s directions unless otherwise indicated in 6.1.
as Test Method F895 may be considered as an alternate
method,dependingonsamplecharacteristics,orincaseswhere
investigators wish to further evaluate the cytotoxic response of 7. Cell Cultures
cells underlying the test sample.
7.1 Cell cultures used in this assay should be the ATCC,
CCL 1 NCTC clone 929 strain (clone of Strain L, mouse
5. Apparatus
connective tissue) designated L-929. Other suitable validated
5.1 The following apparatus shall be used:
cell lines may be considered. Cells should be tested periodi-
cally for Mycoplasma contamination. A passage or doubling
5.2 Incubator, to maintain a temperature of 37 6 1°C with
limit should be established within the laboratory with support-
a humidified atmosphere of 4 to 6% CO in air.
ive data to prevent the use of aged cells in this practice, which
5.3 Tissue Culture Grade Culture Dishes,thataresterileand
may exhibit phenotypic abnormalities that could affect mor-
60 mm in diameter by 10 mm deep. Alternative sizes may be
phologic assessment.
suitable if the grading recommended in Table 1 can be
performed adequately.
8. Control Materials
NOTE 2—Plastic dishes are recommended because they provide a flat
8.1 Prepare negative control specimens in accordance with
surface that contributes to the formation of a uniform cell monolayer.
Section 10 from a material that consistently elicits negligible
5.4 Disposable, Sterile, Centrifuge Tubes.
cellular response in this assay (for example, USP Negative
5.5 Inverted Optical Microscope, with magnifications of Control Plastic Reference Standard).
40×, 100×, and 200×.
8.2 Prepare positive control specimens in accordance with
5.6 Clinical Centrifuge, capable of attaining 1300xg. Section 10 from a material that consistently elicits a
predictable, moderate degree of cytotoxicity.
5.7 Filter Disks—10 mm in diameter (for evaluation of
8.2.1 Use aqueous phenol (0.45 6 0.05% by volume) as a
liquids).
positive control for a diffuse reaction of cellular degeneration
NOTE3—MilliporeAP2501000filterdiskshavebeenfoundsatisfactory
and sloughing. Take care to ensure that the preparation is
foruseincytotoxicityevaluationsbecausetheyelicitnocytopathiceffect.
homogenous.
Other filter disks that do not elicit a cytopathic effect may also be used.
8.2.2 Latex rubber has been used as a positive polymeric
5.8 Water Bath, capable of maintaining a temperature of 37
control for a zone of reactivity.
6 2°C.
NOTE 4—Alaminar flow work area capable of filtering out 99.99% of
9. General Technique
allparticlesgreaterthan0.5µmindiameter,oraclass100cleanroommay
9.1 Useaseptictechniquethroughoutthisassaytominimize
be necessary to prevent contamination of cultures.
microbial contamination.
6. Reagents
NOTE 8—Mouth pipetting should not be employed to transfer cells,
6.1 The following reagents shall be used: medium, or reagents.
6.1.1 Minimum Essential Medium (MEM), prepared with
9.2 Warm all solutions and materials to a temperature of 37
L-glutamineandaugmentedbytheadditionofEarle’ssaltsand
6 2°C before placing in contact with cells.
5–10 % fetal bovine serum.
10. Preparation of Specimens
NOTE 5—Glutamine may be omitted from this formulation in order to
maximize the shelf life of the medium. Prior to use, 5 mLof L-glutamine
10.1 Sterilize all specimens by a method appropriate to the
solution (see 6.1.2) should be added to each 500 mL of MEM.
end use of the device.
NOTE6—OpenedcontainersofMEMmaybestoredatatemperatureof
2 to 8°C for periods of not more than one week.
10.2 Whereadeviceissufficientlysmall(see10.3and10.4)
NOTE 7—Antibiotics, such as penicillin G10,000 I.U./ml and strepto-
to fit into the culture dish leaving an adequate margin of cells
mycin10,000I.U./ml,maybeaddedtothemedium(1mlofantibioticper
100 ml of media) to reduce the incidence of bacterial contamination.This for evaluation, use the entire device as a specimen.
F813 − 20
10.3 Cutlargesolidmaterialsanddevicesincrosssectionto 11.4 Add an appropriate volume of trypsin solution (0.1%)
obtain a flat surface having an area of 100 mm to be placed in totheflaskforthesizeofcellcultureflaskinuse.Forexample,
direct contact with the cell monolayer. 5 6 0.5 mL of trypsin solution may be used for a 150–cm
culture flask.
10.4 Uneven items and tubing or rod-shaped materials and
11.5 Incubate for 5 to 10 min to dissociate the cells, in
devices may be cut into cross sections and placed side to side
accordance with manufacturer’s recommendation.
to provide a flat test surface that covers an area of 100 mm on
the cell monolayer.
11.6 Dilutethesuspensionsufficientlytoreducetheeffectof
the trypsin or transfer the cell suspension to a centrifuge tube
10.5 Materials or devices with irregular or complex surface
andcentrifugeat1300gfor6minanddiscardthesupernatant.
areas such as resins, non-soluble powders, pellets, or molding
with porous or complex shapes may be placed on the cell
11.7 Dilute or suspend the cells in 10 6 0.1 mL of fresh
monolayer so that it covers an area of 100 mm on the cell
medium, and mix the suspension thoroughly.
monolayer as uniformly as possible.
11.8 Add 6.0 6 0.1 mL of medium to each culture dish.
10.6 Obtain specimens from larger medical items from
11.9 Using a sterile 10-mL serological pipette, add 8 to 12
locations with relatively large cross sections in order to expose
drops of cell suspension to each dish.
interior material.
11.10 Incubate until a near-conflu
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: F813 − 07 (Reapproved 2012) F813 − 20
Standard Practice for
Direct Contact Cell Culture Evaluation of Materials for
Medical Devices
This standard is issued under the fixed designation F813; the number immediately following the designation indicates the year of original
adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A superscript
epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
1.1 This practice covers a reference method of direct contact cell culture testing which may be used in evaluating the cytotoxic
potential of materials for use in the construction of medical materials and devices.
1.2 This practice may be used either directly to evaluate materials or as a reference against which other cytotoxicity test methods
may be compared.
1.3 This is one of a series of reference test methods for the assessment of cytotoxic potential, employing different techniques.
1.4 Assessment of cytotoxicity is one of several tests employed in determining the biological response to a material, as
recommended in Practice F748.
1.5 The L-929 cell line was chosen because it has a significant history of use in assays of this type. This is not intended to imply
that its use is preferred; only that the L-929 is a well-characterized, well characterized, readily available, established cell line that
has demonstrated reproducible results in several laboratories.
1.6 Since the test sample is not removed at the time of microscopic evaluation and underlying cells may be affected by the
specific gravity of the test sample, this practice is limited to evaluation of cells outside the perimeter of the overlying test sample.
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use.
1.8 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
F619 Practice for Extraction of Medical Plastics
F748 Practice for Selecting Generic Biological Test Methods for Materials and Devices
F895 Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
F1027 Practice for Assessment of Tissue and Cell Compatibility of Orofacial Prosthetic Materials and Devices
2.2 Other Documents:
The American Type Culture Collection (ATCC), Catalogue of Strains II
USP Negative Control Plastic Reference Standard
This practice is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and Devices and is the direct responsibility of Subcommittee F04.16
on Biocompatibility Test Methods.
Current edition approved Oct. 1, 2012April 1, 2020. Published November 2012June 2020. Originally approved in 2001. Last previous edition approved in 20072012 as
F813 – 07.F813 – 07(2012). DOI: 10.1520/F0813-07R12.10.1520/F0813-20.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
American Type Culture Collection, P.O. Box 1549, Manassas, VA 20108.
U.S. Pharmacopeia, Vol 24, Rand McNally, Taunton, MA, 1994, pp. 1652–1653. Use latest publication to ensure current cumulative revisions are used.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F813 − 20
3. Summary of Practice
3.1 Cell cultures are grown to a confluent monolayer in culture dishes. The growth medium is aspirated and replenished to
provide a resting, confluent cell layer. Test and control specimens are placed in direct contact with the cell layer to provide an
accelerated assessment of the presence or absence of a cytotoxic effect from a given material or device. See Practice F1027 for
definitions.
4. Significance and Use
4.1 This practice is useful for assessing cytotoxic potential both when evaluating new materials or formulations for possible
use in medical applications, and as part of a quality control program for established medical materials and medical devices.
4.2 This practice assumes that assessment of cytotoxicity potential provides one method for predicting the potential for
cytotoxic or necrotic reactions to medical materials and devices during clinical applications to humans. In general, cell culture
testing methods have shown good correlation with animal assays and are frequently more sensitive to toxic moieties.when only
chemical toxicities are being considered.
NOTE 1—The results obtained using this method may not predict in vivo behavior which can be influenced by multiple factors such as those arising
from site of application or physical properties that may result from design and fabrication.
4.3 This cell culture test method is suitable for adoption in specifications and standards for materials for use in the construction
of medical devices that are intended to be implanted in the human body or placed in have direct contact with tissue, tissue fluids,
or blood on a long-term basis. blood. However, care should be taken when testing materials that are resorbable to be sure
absorbable, include an eluting or degradable coating, are liquid or gelatinous in nature, are irregularly shaped solid materials, or
have a high density or mass, to make sure that the method is applicable. If leachables from the test sample are capable of diffusing
through the agar layer, agarose-based methods such as Test Method F895 may be considered as an alternate method, depending
on sample characteristics, or in cases where investigators wish to further evaluate the cytotoxic response of cells underlying the
test sample.
4.4 Since cells in this direct contact test method are not protected by an overlying agarose layer, they are more susceptible to
potential mechanical damage imparted by the overlying test sample. Investigators wishing to evaluate the cytotoxic response of
cells underlying the test sample should consider agarose-based methods similar to Test Method F895. Alternatively, depending on
sample characteristics, extraction methods such as Practice F619 may also be considered.
5. Apparatus
5.1 The following apparatus shall be used:
5.2 Incubator, to maintain a temperature of 37 6 2°C and 1°C with a humidified atmosphere of 4 to 6 % CO with greater than
90 % relative humidity.in air.
5.3 Tissue Culture Grade Culture Dishes, that are sterile and 3560 mm in diameter by 10 mm deep. Alternative sizes may be
suitable if the grading recommended in Table 1 can be performed adequately.
NOTE 2—Plastic dishes are recommended because they provide a flat surface that contributes to the formation of a uniform cell monolayer.
5.4 Disposable, Sterile, Centrifuge Tubes.
5.5 Inverted Optical Microscope, with magnifications of 40×, 100×, and 200×.
5.6 Clinical Centrifuge, capable of attaining 1300xg.
5.7 Filter Disks—10 mm in diameter (for evaluation of liquids).
NOTE 3—Millipore AP2501000 filter disks have been found satisfactory for use in cytotoxicity evaluations because they elicit no cytopathic effect.
Other filter disks that do not elicit a cytopathic effect may also be used.
5.8 Water Bath, capable of maintaining a temperature of 37 6 2°C.
NOTE 4—A laminar flow work area capable of filtering out 99.99 % of all particles greater than 0.5 μm in diameter, or a class 100 clean room may
be necessary to prevent contamination of cultures.
6. Reagents
6.1 The following reagents shall be used:
6.1.1 Minimum Essential Medium (MEM), prepared withoutwith L-glutamine and augmented by the addition of Earle’s salts
and 5–10 % 5–10 % fetal bovine serum.
NOTE 5—Glutamine is may be omitted from this formulation in order to maximize the shelf life of the medium. Immediately beforePrior to use, 5 mL
of L-glutamine solution (see 6.1.2) are should be added to each 500 mL of MEM.
NOTE 6—Opened containers of MEM may be stored at a temperature of 2 to 8°C for periods of not more than one week.
NOTE 7—Antibiotics, such as penicillin G10,000 I.U./ml and streptomycin 10,000 I.U./ml, may be added to the medium (1 ml of antibiotic per 100
ml of media) to reduce the incidence of bacterial contamination. This may, however, have an adverse effect on the viability of the cell cultures.
F813 − 20
6.1.2 L-glutamine Solution, 29.2 mg/mL of sterile water.
6.1.3 Hanks’ Solution, calcium-and magnesium-free (store at room temperature).
6.1.4 Trypsin, 0.1 % solution in Hanks’ solution or calcium- and magnesium-free, phosphate-buffered saline (store frozen).
6.1.5 Water, distilled, deionized, and sterile, with a minimum resistivity of 1 MΩ·cm.
6.2 All reagents shall be tissue-culture grade or equivalent.
6.3 Reagents shall be reconstituted in accordance with the manufacturer’s directions, using aseptic technique.
6.4 Reagents shall be stored in accordance with the manufacturer’s directions unless otherwise indicated in 6.1.
7. Cell Cultures
7.1 Cell cultures used in this assay should be the ATCC, CCL 1 NCTC clone 929 strain (clone of Strain L, mouse connective
tissue) designated L-929. Other suitable validated cell lines may be considered. Cells should be tested periodically for Mycoplasma
contamination. A passage or doubling limit should be established within the laboratory with supportive data to prevent the use of
aged cells in this practice, which may exhibit phenotypic abnormalities that could affect morphologic assessment.
8. Control Materials
8.1 Prepare negative control specimens in accordance with Section 10 from a material that consistently elicits negligible cellular
response in this assay (for example, USP Negative Control Plastic Reference Standard).
8.2 Prepare positive control specimens in accordance with Section 10 from a material that consistently elicits a predictable,
moderate degree of cytotoxicity.
8.2.1 Use aqueous phenol (0.45 6 0.05 % by volume) as a positive control for a diffuse reaction of cellular degeneration and
sloughing. Take care to ensure that the preparation is homogenous.
8.2.2 Latex rubber has been used as a positive polymeric control for a zone of inhibition.reactivity.
9. General Technique
9.1 Use the aseptic technique throughout this assay to minimize microbial contamination.
NOTE 8—Mouth pipetting should not be employed to transfer cells, medium, or reagents.
9.2 Warm all solutions and materials to a temperature of 37 6 2°C before placing in contact with cells.
10. Preparation of Specimens
10.1 Sterilize all specimens by a method appropriate to the end use of the device.
10.2 Where a device is sufficiently small (see 10.3 and 10.4) to fit into the culture dish leaving an adequate margin of cells for
evaluation, use the entire device as a specimen.
10.3 Cut large solid materials and devices in cross section to obtain a flat surface having an area of 100 to 250 mm to be placed
in direct contact with the cell monolayer.
10.4 Uneven items and tubing or rod-shaped materials and devices may be cut into cross sections and placed side to side to
provide a flat test surface that covers an area of 100 mm on the cell monolayer.
10.5 Prepare specimens of rod orMaterials or devices with irregular or complex surface areas such as resins, non-soluble
powders, pellets, or molding with porous or complex shapes may be placed on the cell monolayer so that it covers an area of 100
mm tubing or of rod- or tube-shaped devices as follows:on the cell monolayer as uniformly as possible.
10.4.1 Where the diameter is less than 6.4 mm, cut 5 to 15 mm in length.
10.4.2 Where the diameter is 6.4 to 15 mm, cut 2 to 8 mm in length.
10.4.3 Where the diameter exceeds 15 mm, prepare cross-sections as described in 10.3.
10.6 Obtain specimens from larger medical items from locations with relatively large cross sections in order to expose interior
material.
10.7 If a device is constructed of two or more materials, cut either the test specimen from the materials’ interface or test separate
specimens from each material.
10.8 Prepare specimens for evaluating the cytotoxicity of liquids or extracts liquids, including negative and positive controls,
by saturating a sterile filter disk (see 5.7) and allowing the excess liquid to drain off while maintaining asepsis. Use the saturated
filter disk as a test specimen.
NOTE 9—When ethylene oxide or other chemical sterilants are used, adequate aeration time should be allowed, to permit dissipation of residues which
may adversely affect the results recorded in this assay. The aeration time should be based on the time established by the sterilization validation procedure
for the dissipation of the residues.
NOTE 10—In general, the specimens should be cleaned to remove any residues from specimen preparation, and sterilized after they have been cut to
size. prepared following aseptic technique. If the large solid materials are very hard, like ceramics,hard (like ceramics) which require cutting with metal
F813 − 20
or diamond saws, care should be taken to remove any contamination from the metal blade or from the metal bonding the diamonds to the blade. When
evaluating the cytotoxicity potential of medical materials or devices that are contained in the final sterile package, resterilization, further processing, or
delay between the time of opening the package and starting the test must be avoided. With small items the entire content of the sterile package may be
used as th
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